Humoral responses to blood-borne pathogens are mediated by a specialized subset of splenic B cells known as marginal zone (MZ) B cells. These cells, which are strategically positioned to encounter foreign antigen as it enters the spleen, rapidly produce an effective antibody response and, therefore, represent a first line of defense against blood-borne pathogens. While it is known that MZ B cell development and function are dependent on signaling through a variety of surface receptors including the B cell antigen receptor (BCR), Notch, the tumor necrosis factor receptor (TNFR) family, the G-protein-coupled receptor (GPCR) family, the toll-like receptor (TLR) family, and the type 1 interferon receptor (IFNR) family, it is still not understood how and when these diverse signaling pathways interact to generate functional MZ B cells. In a recently published study, we reported that Blk, a B cell-specific member of the Src family of tyrosine kinases (SFKs), is differentially expressed in mature splenic B cell subsets, with MZ B cells expressing twice as much Blk as follicular (FO) B cells. Importantly, this difference in Blk expression levels reflected a differential requirement fr Blk in MZ and FO B cell development, as both Blk-deficient and Blk-haploinsufficient mice exhibited defects in the development of MZ but not FO B cells. Moreover, although there were fewer MZ B cells in Blk+/- and Blk-/- mice compared to Blk+/+ mice, Blk mutant MZ B cells were hyper-responsive to BCR stimulation, both in vitro and in vivo. Together, these data demonstrate a previously undocumented role for Blk in the development and activation of MZ B cells. Our long-term goal is to determine the mechanism(s) by which high levels of Blk control MZ B cell development and function. Since SFKs participate in a multitude of signaling pathways, we hypothesize that increasing Blk levels improves its utilization by different classes of surface receptors, thereby providing a mechanism for communication among the diverse signaling pathways that contribute to the generation and maintenance of MZ B cells. The objective of this R21 grant application is to test this novel hypothesis by determining which signaling pathways upregulate Blk expression during MZ B cell development and which require Blk activity during MZ B cell development and activation. We expect that the proposed study will not only determine whether Blk is a key component in the BCR, Notch, GPCR, TNFR, TLR and type IFNR signaling pathways but will also shed light on how and when these diverse signaling pathways interact during MZ B cell development and activation. This study is important because, by understanding the molecular requirements for their proper development and function, it will facilitate development of vaccines and therapies that specifically target MZ B cells.